One of the primary functions of the human immune system is to elicit responses that protect against infections by controlling the spread and virulence of invading pathogens. Central to maintaining immunity is the cell-surface presentation of antigenic peptides to CD8+ cytotoxic T-lymphocytes by class I major histocompatibility complex (MHC) molecules. It is now well-established that viruses have evolved selective mechanisms to subvert the host antiviral cellular immune defenses and establish persistent infections. In most cases, these mechanisms operate by the action of viral proteins able to suppress the expression of class I MHC molecules at the cell-surface. This proposal is focused on investigating the immunomodulatory function of one such viral protein from Adenoviruses (Ads). The Ad genome includes a number of genes that code for immunomodulatory proteins, of which the 19 kDa protein from E3 (E3-19K) is the best characterized. E3-19K is a type I membrane protein that has been shown to associate with class I MHC molecules in the endoplasmic reticulum (ER) of Ad-infected cells, and prevent their transport to the cell-surface. Two structural features of E3-19K are thought to be responsible for these observations: its lumenal domain associates with the lumenal domain of class I MHC molecules, whereas its C-terminal cytosolic tail functions to retain class I MHC molecules in the ER by virtue of an ER-retrieval motif. Several molecular and mechanistic aspects of this process are poorly understood. This Research Plan has two specific goals; to produce recombinant, soluble E3-19K and characterize its biochemical and biophysical properties; and to reconstitute in vitro its interactions with two distinct forms, peptide-filled and "empty", of recombinant, soluble class I MHC molecules. These studies will provide insights in the region of E3-19K that binds to class I MHC molecules, as well as direct evidence to suggest how E3-19K may function in relation to the sequential series of events in the class I assembly pathway. This knowledge will increase our understanding of Ad pathogenesis and host cell biology. This is fundamental to our ability to prevent, treat, and cure, Ad-induced immune dysfunctions associated with respiratory, gastrointestinal, ocular, and urinary tract diseases. These studies are also important in relation to immunization and gene therapy given the potential to use Ads as vectors for the delivery of genes.